Project Summary/Abstract We have made the seminal observation that O-linked -N-acetylglucosamine (O-GlcNAc) modification of proteins induced by three distinct and independent stimuli, 17- estradiol (E2), glucosamine (GlcN) and the selective O-GlcNAcase inhibitor O-(acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc), has anti-inflammatory effects in balloon injured rat carotid arteries. These novel observations provide provocative evidence that enhanced O-GlcNAc modification of proteins has anti-inflammatory and vasoprotective effects in the setting of acute endoluminal vascular injury. We have strong evidence that increased protein O-GlcNAc modification in response to GlcN treatment is associated with attenuation of TNF- -induced expression of inflammatory mediators in isolated rat aortic smooth muscle cells (RASMCs) in a manner previously reported for E2. We have utilized the TNF--treated RASMC as an in vitro model of the acute vascular injury response and have begun to define the mechanisms by which interventions that stimulate protein O-GlcNAc modification, i.e., E2 and GlcN, inhibit inflammatory responses to TNF-. We have focused on the NFB signaling pathway, which is known to be activated by both TNF- and acute vascular injury. Initial experiments demonstrated that GlcN inhibits TNF--induced NFB activation in RASMCs. Subsequent studies showed that pretreatment with GlcN inhibits TNF--induced phosphorylation and degradation of IB in RASMCs, while E2 pretreatment is associated with an initial reduction, followed by an accelerated reappearance of IB in TNF- treated cells, likely reflecting new protein synthesis mediated by activated NFB. The current study will test directly the hypothesis that O-GlcNAc modification of proteins, including IB, plays a mechanistic role in regulating the inflammatory response to endoluminal arterial injury in vivo and to TNF- stimulation in isolated RASMCs in vitro. The Specific Aims are: Specific Aim 1: To test the hypothesis that increasing protein O-GlcNAc modification protects arteries from inflammatory stress related to acute endoluminal injury in vivo via inhibition of NFB signaling. Specific Aim 2: To test the hypothesis that increasing protein O-GlcNAc modification inhibits TNF--induced inflammatory responses in RASMCs in vitro via inhibition of NFB signaling and define the precise sites in the NFB signaling cascade that are responsible for this effect. Specific Aim 3: To identify specific protein targets of O-GlcNAc modification in RASMCs that play a functional role in the anti-inflammatory effects of GlcN and E2. Upon successful completion of these Aims, cellular/molecular mechanisms responsible for the anti-inflammatory and vasoprotective actions of O- GlcNAc modification will be elucidated and will be related to the extent of the injury response (i.e., inflammation and neointima formation). We postulate that O-GlcNAc modification represents a novel mechanism of vasoprotection that may lend itself to the development of new strategies for the prevention and treatment of cardiovascular disease.